A new contribution to the raptorial ciliate genus Lacrymaria (Protista: Ciliophora): a brief review and comprehensive descriptions of two new species from Changjiang Estuary

Ciliates serve as excellent indicators for water quality monitoring. However, their utilization is hindered by various taxonomic confusions. The ciliate genus Lacrymaria Bory de Saint-Vincent, 1824 is commonly found in different aquatic habitats, but its taxonomy has been sparsely investigated using state-of-the-art methods. This study investigated two new Lacrymaria species from Nanhui Wetland, Shanghai, China, using living observation, protargol staining, and molecular phylogeny methods. Lacrymaria songi sp. nov. is 180–340 × 20–25 μm in size and possesses 12–16 somatic kineties, 1 terminal contractile vacuole, 2 macronuclear nodules, and 2 types of rod-shaped extrusomes. Lacrymaria dragescoi sp. nov. is distinguished from its congeners by its cell size of 210–400 × 25–35 μm, 14–17 somatic kineties, 1 terminal contractile vacuole, 1 macronucleus, and 2 types of rod-shaped extrusomes. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Lacrymariidae is monophyletic but Lacrymaria is not. Additionally, a brief review of the genus Lacrymaria is provided in this study. We suggest that L. bulbosa Alekperov, 1984, L. lanceolata Kahl, 1930, and L. ovata Burkovsky, 1970 be removed from the genus and propose Phialina lanceolata nov. comb. and Phialina ovata nov. comb. for the latter two. ZooBank registration: Present work: urn:lsid:zoobank.org:pub:CDFB1EBD-80BD-4533-B391-CEE89F62EDC4 Lacrymaria songi sp. nov.: urn:lsid:zoobank.org:act:417E7C2D-DAEC-4711-90BB-64AB3CD2F7D5 Lacrymaria dragescoi sp. nov.: urn:lsid:zoobank.org:act:8778D6B0-1F2E-473C-BE19-3F685391A40D.

Recent studies on haptorian ciliates in China have revealed a high diversity of the order Pleurostomatida (Liu et al., 2017;Pan et al., 2020;Wu et al., 2021Wu et al., , 2022;;Zhang G. et al., 2022aZhang G. et al., ,b,c, 2023)).However, little attention has been given to the family Lacrymariidae.A project on ciliate fauna conducted in Changjiang Estuary has led to the discovery of various new or rarely known ciliates (Chen et al., 2022;Han et al., 2022;He et al., 2022;Zhang Z. et al., 2022a,b).As a new contribution, we investigated the phylogeny and taxonomy of two new Lacrymaria species, namely, L. songi sp.nov.and L. dragescoi sp.nov., using integrative methods including live observation, silver staining, and DNA sequencing.Additionally, we provide a brief review of the genus Lacrymaria.

Sample collection and cultivation
Lacrymaria songi sp.nov.and Lacrymaria dragescoi sp.nov.were both collected on 28 September 2022 from two adjacent sites of Nanhui Wetland (N30°53′27.56″,E121°58′38.78″),Shanghai, China (Figure 1).For the habitat of L. songi sp.nov., the water temperature was 23.6°C, the pH was 7.43, the concentration of dissolved oxygen (DO) was 4.22 mg/L, and the salinity measured in the Petri dish was 17‰; for the habitat of L. dragescoi sp.nov., the water temperature was 23.6°C, the pH was 7.61, the DO was 5.47 mg/L, and the salinity measured in the Petri dish was 20‰.All environmental parameters, except salinity, were measured in situ, and the salinity was measured when Lacrymaria species were detected in the raw cultures.
After the samples were transported to the laboratory, raw cultures were immediately established in Petri dishes with rice grains to enrich the growth of bacteria, serving as food for the ciliates.L. songi sp.nov.and L. dragescoi sp.nov.were detected after 3 weeks.

Morphological observation
Live cells were observed using bright field and differential interference contrast microscopy (Olympus BX53) at magnifications of 100-1,000.The ciliary pattern was revealed using the protargol preparation method (Wilbert, 1975).The protargol reagent was manually synthesized following the method described by Pan et al. (2013).Counts and measurements of stained specimens were performed at a magnification of 1,000, and drawings were made at the same magnification with the aid of a camera lucida.Terminology and systematics are explained by Lynn (2008) andVd'ačný et al. (2011).

DNA extraction, PCR amplification, and DNA sequencing
Five cells of each species were isolated from the raw cultures using sterile micropipettes and washed at least five times with filtered (0.22 μm) habitat water to remove contaminants.Genomic DNA was extracted by a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) using one-quarter of the volume recommended by the manufacturer's instructions as described by Shao et al. (2023).The SSU rRNA gene was amplified by PCR using the primers 18S-F (5'-AAC CTG GTT GAT CCT GCC AGT-3′) and 5.8S-R (5′-TAC TGA TAT GCT TAA GTT CAG CGG-3′) (Medlin et al., 1988;Sogin, 1989).The cycling parameters were as follows: an initial denaturation of 3 min at 95°C, followed by 30 cycles of 30 s at 95°C, 20 s at 56°C, and 1.5 min at 72°C, with a final extension of 5 min at 72°C.The PCR products were, then, purified, cloned, and sequenced, following the method described by Chen et al. (2022).The sequencing data were assembled using SeqMan v7.1 (DNAStar), and sequence similarities were calculated using BioEdit v.7.2.5 (Hall, 1999).
The support of the dataset for competing phylogenetic hypotheses was evaluated using the approximately unbiased (AU) test to test the monophyly of the genus Lacrymaria (Shimodaira and Hasegawa, 2001).
The site-wise likelihoods for the resulting constrained topology and the non-constrained ML topology were calculated using RAxML v.8.2.11 under a partitioned GTR + GAMMA model (Yang, 1996;Stamatakis, 2014).The same model was used to estimate the site likelihoods for those trees prior to conducting the AU test.The scores of each constraint tree were compared with the unconstrained ML result using the AU test option implemented in CONSEL (Shimodaira and Hasegawa, 2001).

Diagnosis
Size: approximately 180-340 × 20-25 μm in vivo.Body shape: highly variable depending on the state of contraction, ranging from a vase-shaped body in the contracted state to fusiform to clavate in the extended state.Neck: flexible, occupying half of the body length and up to two-thirds of body length when swimming, and neck beating 92 times/min when preying.Extrusomes have two types: type I-approximately 10 μm long, rod-shaped, mostly arranged in bundles, scattered in main body, and attached to oral bulge; type II-approximately 4 μm long, rod-shaped, scattered in main body and 12-16 somatic kineties.Single terminally located contractile vacuole.Two macronuclear nodules.Brackish habitat.

Type specimens
A protargol slide (registration no.TJ2022090805-1) with the holotype circled in black ink and one paratype slide (TJ2022090805-2) are deposited in the Laboratory of Protozoology, Ocean University of China.

Dedication
The species is named in honor of Prof. Weibo Song, Ocean University of China, in recognition of his outstanding contribution to Ciliatology.The SSU rRNA gene sequence of L. songi sp.nov.has been deposited in GenBank (accession no.OR689566) with 1,641 bp long and GC content of 42.41%.

Description
Cell: highly contractile, when fully extended cell size in vivo approximately 180-340 × 20-25 μm and length:width ratio of 11:1 (Figures 2A, 3A-C) and when contracted, cell size approximately 65-102 × 28-40 μm and length:width ratio of 3:1 (Figures 2D, 3D).Body shape fusiform to clavate with flexible neck, occupying half of body length, and up to two-thirds of body length when swimming.The posterior end tapered and tail-like when free swimming but vaseshaped with neck retracting into trunk and the posterior end broadly tapered when contracted (Figures 2A,D,H, 3A,E,J,K).
Two ovoidal macronuclear nodules centrally located with a filament connected to each other, each approximately 12-20 × 8-13 μm in vivo and approximately 10-32 × 6-21 μm after protargol staining (Figures 2G, 3I,M and Table 1).Two micronuclei detected only in 1 out of 30 stained individuals, respectively, located at subapical of each macronuclear nodule (Figure 2G, 3M).However, micronucleus not detected in live cells.Single contractile vacuole terminally located, variable in shape, ranging from rounded to obovate, approximately 11 × 17 μm during diastole, pulsating every 5 min (Figure 3E).Two types of extrusomes: type I approximately 10 μm long, rod-shaped, straight or slightly curved, mostly arranged in bundles, scattered in main body, and attached to oral bulge; type II approximately 4 μm long, rod-shaped, straight or slightly curved, scattered in main body (Figures 2C,F, 3G,H,L,P).Both types of extrusomes easily detected after protargol staining but only type I detectable in vivo.Two types of colorless cortical granules: type I dot-like, approximately 0.4 μm in vivo, densely arranged in five or six rows between kineties in peripheral region of cortex, this character may vary slightly with body contraction; type II dot-like to oval-shaped, approximately 0.8 μm in vivo, only distributed along somatic kineties deep in cortex (Figures 2B,E,  3N,O).Cytoplasm colorless or grayish, containing numerous globular granules (<4 μm in diameter) in trunk, rendering neck hyaline and trunk opaque (Figure 3L).Locomotion usually by swimming fast with neck swinging; when preying, neck extends forward and backward and retracts rapidly, beating approximately 92 times per minute, whereas trunk moves in a small range (Supplementary Video S1).

Diagnosis
Size: approximately 210-400 × 25-35 μm in vivo.Body shape: highly variable depending on the state of contraction, ranging from a vase-shaped body in the contracted state to fusiform to clavate in the extended state.Neck: flexible, occupying half of the body length, and accounting for two-thirds of the body length when swimming, and neck beating 30 times/min when preying.Extrusomes have two types; type I-approximately 13 μm long, rod-shaped, mostly arranged in bundles, scattered in main body and attached to oral bulge; type II-approximately 3 μm long, rod-shaped, scattered in main body, and 14-17 somatic kineties.Single terminally located contractile vacuole.One macronuclear nodule.Brackish habitat.

Type specimens
A protargol slide (registration no.TJ2022090807-1) with the holotype circled in black ink and one paratype slide (TJ2022090807-2) are deposited in the Laboratory of Protozoology, Ocean University of China, Qingdao, Shandong, China.

Dedication
The species is named in honor of Prof. Jean Dragesco, in recognition of his contributions to Ciliatology.

SSU rRNA gene sequence
The SSU rRNA gene sequence of L. dragescoi sp.nov.has been deposited in GenBank (accession no.OR689567) with 1,642 bp long and GC content of 42.75%.4A, 5A,B); when contracted, cell size of approximately 100-170 × 36-44 μm and length:width ratio of 3:1 (Figures 4C, 5E).Body shape fusiform to clavate with flexible neck, occupying half of the body length and accounting for two-thirds of the body length when swimming.The posterior end tapered and tail-like when free swimming but vase-shaped with neck retracting into trunk, and posterior end sharply rounded when contracted (Figures 4A-C,  5A,D,E).
Nuclear apparatus centrally located, comprising one ovalshaped macronucleus, approximately 17-25 × 6-9 μm in vivo and approximately 20-40 × 7-24 μm after protargol staining (Figures 4H,  5H,I and Table 1).Micronucleus undetected in vivo or after protargol staining.Single contractile vacuole terminally located, variable in shape, ranging from rounded to obovate, approximately 17 × 12 μm during diastole, and pulsating every 3 min (Figures 4A,  5A).Two types of extrusomes: type I approximately 13 μm long, rod-shaped, straight or slightly curved, mostly arranged in bundles, scattered in main body, and attached to oral bulge; type II approximately 3 μm in size, rod-shaped, straight or slightly curved, scattered in main body (Figures 4D,F,H, 5H,I).Both types of extrusomes easily detected after protargol staining but only type I detectable in vivo.Two types of cortical granules: type I dot-like, approximately 0.4 μm in vivo, in the peripheral region of cortex densely arranged in seven or eight rows between kineties, this character may vary slightly with body contraction; type II dot-like to oval-shaped, approximately 0.7 μm in vivo, only distributed along somatic kineties deep in cortex; both types of cortical granules colorless ( Figures 4E,G, 5F,G).Cytoplasm colorless or grayish, containing numerous globular granules (< 2.6 μm in diameter) in trunk, rendering neck hyaline and trunk opaque (Figure 5H).Locomotion usually by swimming fast with neck swinging; when preying, neck extends forward and and retracts rapidly, beating approximately 30 times per minute, whereas trunk moves in a small range (Supplementary Video S2).

Sequence comparison and molecular phylogeny
The nucleotide similarities of the SSU rRNA gene sequences between Lacrymaria species range from 94.45 to 99.62%.L. songi sp.nov.differs from congeners except for L. dragescoi sp.nov.by 25-66 nucleotides, with sequence identities ranging from 95.69 to 98.43%.L. dragescoi sp.nov.differs from congeners except for L. songi sp.nov.by 6-60 nucleotides, with sequence identities ranging from 96.08 to 99.62% (Figure 6).
The topologies of the ML and BI trees were basically congruent with varying levels of support; therefore, only the ML tree is presented in Figure 7.As shown in the ML tree, Lacrymaria songi sp.nov.and L. dragescoi sp.nov.fall in the core of Lacrymaria, and the family Lacrymariidae is recovered as a monophyletic group (Figure 7).The genus Lacrymaria is non-monophyletic with Lacrymaria sp.(MF474345) groups with Phialina.The AU test also refutes the monophyly of Lacrymaria (AU > 0.05).In the ML tree, L. dragescoi sp.nov.groups with Lacrymaria marina pop1 (MF474343) with high support (ML/BI, 100%/0.99),forming a sister clade to L. songi sp.nov.Then, they depict a monophyletic group that is sister to a clade formed with very weak support by Lacrymaria olor clone 1-5 (MN30553-MN30557) and Lacrymariidae (LN869967).Lacrymaria is easily recognized by its long, contractile, and flexible neck.However, the history of Lacrymaria is marked by confusion.Both Lacrymaria and its relative Phialina were originally defined based on misinterpreted oral features.Phialina has also experienced abandonment and re-activation (Bory de Saint-Vincent, 1824;Foissner, 1983).Currently, there are approximately 53 nominal species within the genus Lacrymaria (Supplementary Table S1).However, only 12 species have been investigated through live observation and silver Furthermore, those Lacrymaria species without infraciliature data have mostly not been rediscovered since their original reports raised questions about their validation and affiliations.Lacrymaria bulbosa Alekperov, 1984, L. lanceolata Kahl, 1930, and L. ovata Burkovsky, 1970 do not possess a contractile neck (Kahl, 1930;Gelei, 1954;Burkovsky, 1970a;Alekperov, 1984).This indicates that they should be removed from Lacrymaria.There are three genera of Lacrymaria with an acontractile neck, namely, Pelagolacrymaria Foissner et al., 1999, Phialina Bory de St. Vincent, 1824, and Phialinides Foissner, 1988.In terms of ciliary patterns, L. lanceolata and L. ovata lack a monokinetid circle and a dikinetid circle between the head and the trunk.Therefore, they should be assigned to the genus Phialina as new combinations, i.e., Phialina lanceolata nov.comb.and Phialina ovata nov.comb.However, the affiliation of L. bulbosa cannot be determined presently due to its unknown ciliary pattern.

Discussion
Lacrymaria sapropelica Kahl, 1927 andL. urnula Kahl, 1930 both have a furrow encircling the neck-like region, which indicates that they should belong to the family Lagynusidae (Jiang et al., 2023).Since their ciliary patterns have not been investigated, further investigation is needed to determine their exact affiliations, particularly through protargol staining and SSU rRNA gene sequencing.
The molecular phylogeny of Lacrymaria was initially investigated by sequencing the SSU rRNA gene of L. marina Kahl, 1933(Gao et al., 2008).Subsequently, Rossi et al. (2016), Huang et al. (2018), andRajter et al. (2019) sequenced nine new SSU rRNA gene sequences of the genus and found that Lacrymaria was likely a non-monophyletic genus.However, none of those sequences were reported with the morphological data, which cast doubt on these results.

Comments on Lacrymaria songi sp. nov.
Previous studies indicate that the following characteristics can be used for the circumscription of Lacrymaria species: the number of somatic kineties, the number of macronuclear nodules, the number and position of micronuclei, the number and position of contractile vacuoles, and characteristics of the extrusomes (Foissner, 1983;Dragesco and Dragesco-Kernéis, 1986;Song and Wilbert, 1989;Foissner et al., 1995;Rajter et al., 2019;Wang et al., 2019).
Lacrymaria olor (Müller, 1786) Bory de Saint-Vincent, 1824 resembles L. songi sp.nov. in body size, the number of somatic kineties, and the shape of the posterior end (Foissner et al., 1995).However, L. olor can be clearly distinguished from L. songi sp.nov by the location of the micronucleus (a micronucleus located between the two macronuclear nodules vs. two micronuclei located at the subapical of each macronuclear nodule) and habitat (freshwater vs. brackish water).

FIGURE 1
FIGURE 1 Sampling sites (A-D).(A) A part of the map of China showing the location of Shanghai.(B) A satellite image of Shanghai showing the location of Nanhui Wetland.(C) Photograph of the sampling site in a tidal flat of Nanhui Wetland (N30°53′27.56″,E121°58′38.78″) from where Lacrymaria songi sp.nov.was collected.(D) Photograph of the sampling site in the tidal flat of Nanhui Wetland (N30°53′5.39″,E121°53′37.03″) from where Lacrymaria dragescoi sp.nov.was collected.
FIGURE 2 (A-H) Morphology of Lacrymaria songi sp.nov.from life (A,B,D,E,H) and after protargol staining (C,F,H).(A) Typical extended individual arrow points to the contractile vacuole.(B) Two types of cortical granules; type I is marked by an arrowhead and type II is marked by an arrow.(C) Two types of extrusomes.(D) Shape variants in one individual.(E) Arrangement of cortical granules on the cell surface; type I is marked by an arrowhead and type II is marked by an arrow.(F) The anterior end of the ciliary pattern.(G) Ciliary pattern of the holotype specimen; arrowhead points to a long type of extrusome, arrow points to a short type of extrusome, and red dots indicate micronuclei.(H) Shape variants in different individuals.Scale bars: 100 μm in (A,H); 5 μm in (C); 50 μm in (D,G).

FIGURE 3
FIGURE 3 Photomicrographs of Lacrymaria songi sp.nov.from life (A-E,I-K,M-O) and after protargol staining (F-H,L).(A) A representative individual.(B,C) Different free-swimming individuals show shape variants.(D) A completely contracted individual.(E,J,K) Individuals in different contraction states; arrow in (E) points to the contractile vacuole.(F) Details of the anterior portion showing the anterior somatic kineties.(G) Details of the cytoplasm; arrowhead points to a short extrusome.(H) Details of the cytoplasm; arrowhead points to long extrusomes.(I) A representative specimen showing ciliature and nuclear apparatus.(L) Details of the cytoplasm; arrow points to extrusomes.(M) Details of the cytoplasm; arrowheads point to micronuclei.(N,O) Details of cytoplasm in the middle of the body; arrows point to two different cortical granules.(P) Head structure arrows point to extrusomes.Ma, macronuclear nodules.Scale bars: 100 μm in (A-C); 30 μm in (D); 50 μm in (E,H,J); 23 μm in (I); 12 μm in (F).
4.1.A brief review of the genus LacrymariaBory de Saint-Vincent, 1824 FIGURE 4 (A-H) Morphology of Lacrymaria dragescoi sp.nov.from life (A-C,E,G) and after protargol staining (D,F,H).(A) A typical extended individual; arrow points to the contractile vacuole.(B) Shape variants between different individuals.(C) Shape variants of the same individual.(D) Two types of extrusomes.(E) Two types of cortical granules; type I is indicated by red arrow and type II is indicated by red arrowheads.(F) Anterior end of ciliary pattern showing the head kineties and somatic kineties.(G) Arrangements of the cortical granules on the cell surface; type I is indicated by red arrow and type II is indicated by red arrowheads.(H) Ciliary pattern of the holotype specimen; arrowhead points to the long type of extrusomes and arrow points to the short type of extrusomes.Scale bars: 100 μm in (A,B); 50 μm in (C); 5 μm in (D); 25 μm in (H).

FIGURE 5
FIGURE 5 Photomicrographs of Lacrymaria dragescoi sp.nov.from life (A-H) and after protargol staining (I).(A) A representative individual; arrow points to the contractile vacuole.(B) An extended individual.(C) An individual that is seeking prey; arrow points to a large food vacuole.(D) An individual with a neck somewhat extended for searching for food.(E) A completely contracted individual, arrowhead infers to the head.(F,G) Details of the cell surface in the middle of the body; arrows point to the two different cortical granules.(H) Details of the cytoplasm; arrows point to extrusomes.(I) The holotype specimen showing the ciliature and nuclear apparatus.Ma, macronucleus.Scale bars: 100 μm in (A-D); 50 μm in (E); 30 μm in (I).

TABLE 1
Morphometric characteristics of Lacrymaria songi sp.nov.(the upper line) and L. dragescoi sp.nov.(the lower line) based on protargol stained specimens.a a CV, coefficient of variation (%); M, median; Ma, macronuclear nodule; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of individuals investigated; SD, standard deviation.b Data for Lacrymaria songi sp.nov.are from the anterior macronuclear nodule.